Trank1

Immunohistsochemical analyses were performed as previously described [15 (link)]. Brain sections were deparaffinized through graded alcohols, subjected to heat-induced epitope retrieval for 15 min in 0.01 mol/L citrate buffer (pH 6.0) and incubated for 2 h at room temperature in PBS-albumin blocking serum buffer containing antibodies raised against oxidized low-density lipoprotein (lectine-like) receptors (ORLs) (1:100, Novusbio), leucocyte receptor cluster member 8 (Leng8) (1:200 Novusbio), vomoronasal receptor-1 (Vnr1) (1:300 Abcam), and tetratricopeptide repeat and ankyrin repeat containing1 (Trank1) (1:100 Novusbio). Sections were then incubated for 15 min at room temperature with specific biotinylated secondary antibody and, after several washes in PBS, for 15 min in horseradish peroxidase-avidin/biotin complex solution. Horseradish peroxidase was visualized using 3,3-diaminobenzidinetetrahydrochloride hydrate (DAB, Sigma-Aldrich) and H₂O₂. Counterstaining with hematoxylin-eosin allowed visualization of cell morphology and nuclei by light microscopy.

Western blot analysis was performed according to the standard procedure, using the following antibodies: oxidized low-density lipoprotein (lectine-like) receptors (ORLs) (1:1000, Novusbio), leucocyte receptor cluster member 8 (Leng8) (1:2000 Novusbio), vomoronasal receptor-1 (Vnr1) (1:2000 Abcam), tetratricopeptide repeat and ankyrin repeat containing1 (Trank1) (1:1000 Novusbio), matrix metalloproteinase 9 (MMP9) (1:1000 Abcam), matrix metalloproteinase 2 (MMP2) (1:1000 Abcam), occludin 1 (1:1000, Abcam), plasmalemmal vesicle associated protein-1 (PV-1) (1:2000 Abcam), IL-6 (1:2000 Santa Cruz, CA, USA), anti-TNF (1:600 Santa Cruz, CA, USA), anti-IL-1 beta (1:4000 Santa Cruz, CA, USA), anti-IL-10 (1:2000 Peprotec, London, UK), and beta-actin (1:4000, Sigma-Aldrich). Fifty micrograms of proteins per lane were diluted in loading buffer and denatured at 70 °C for 10 min. Optical densities of the bands were measured using ImageJ software (http://rsb.info.nih.gov/ij/) and normalized against beta-actin.